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ISSN / EISSN : 0749-503X / 1097-0061
Published by: Wiley-Blackwell (10.1002)
Total articles ≅ 4,408
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Gang Luo, Jiying Liu, Ting Bian, Zhendong Zhang,
Published: 6 September 2021
Our previous study revealed that N-acetyl-L-cysteine (NAC) could enhance the secretion of recombinant proteins by Pichia pastoris, but the corresponding molecular mechanisms are still unclear. In the present study, we explored whether other thiols have a similar action on the secretion of recombinant human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ), to reveal the mechanism of NAC on HSA-pFSHβ secretion. Transcriptome analysis showed that genes involved in oxidoreductase activity and oxidation-reduction process were upregulated in cells supplemented with NAC. The other three thiol-reducing regents including dimercaptopropanol (DT), thioglycolic acid, and mercaptolactic acid could improve HSA-pFSHβ production in the culture supernatant. Among them, only DT had similar effect as NAC on HSA-pFSHβ secretion and the increase of GSH content. Moreover, 1-20 mM GSH, 1-10 mM cysteine, or 1-20 mM N-acetyl-D-cysteine supplementation could improve the secretion of HSA-pFSHβ. Furthermore, 0.4-3.2 mM ethacrynic acid, rather than 1-16 mM BSO could inhibit the effect of NAC on the production of HSA-pFSHβ. These results indicated that NAC improved the secretion of HSA-pFSHβ by increasing the intracellular GSH content through its thiol activity rather than as a precursor for GSH synthesis. In conclusion, our results demonstrate, for the first time, that the secretion of recombinant HSA-pFSHβ in Pichia pastoris could be improved through thiol-reducing agent supplementation, and the mechanism of the effect NAC has on HSA-pFSHβ secretion is associated with improving the intracellular GSH content.
, Fernando Carlos Pagnocca,
Published: 2 September 2021
Insects interact with a wide variety of yeasts, often providing a suitable substrate for their growth. Some yeast-insect interactions are tractable models for understanding the relationships between the symbionts. Attine ants are prominent insects on the Neotropics and have performed an ancient fungiculture of mutualistic basidiomycete fungi for more than 55-65 million years. Yeasts gain access to this sophisticated mutualism, prompting diversity, ecological, and biotechnological studies in this environment. We reviewed half a century research in this field, surveying for recurrent yeast taxa and their putative ecological roles in this environment. We found that previous studies mainly covered the yeast diversity from a small fraction of attine ants, being Saccharomycetales, Tremellales, and Trichosporonales as the most frequent yeast or yeast-like orders. Apiotrichum, Aureobasidium, Candida, Cutaneotrichosporon, Debaryomyces, Meyerozyma, Papiliotrema, Rhodotorula, Trichomonascus, and Trichosporon are the most frequent recovered genera. On the other hand, yeast’s ecological role on attine ant-fungus mutualism only tapped the tip of the iceberg. Previous established hypotheses in the literature cover the production of lignocellulosic enzymes, chemical detoxification, and fungus garden protection. Some of these roles have parallels in biotechnological processes. In conclusion, the attine ant environment has a hidden potential for studying yeast biodiversity, ecology, and biotechnology, which has been particularly unexplored considering the vast diversity of fungus-growing ants.
Published: 31 August 2021
Protein tagging is an effective method for characterizing a gene of interest. Tagging can be accomplished in vivo in Saccharomyces cerevisiae by chromosomal integration of a PCR-amplified cassette. However, common tagging cassettes are not suitable for in situ N-terminal tagging when we aim to preserve the gene's endogenous promoter. Existing methods require either two rounds of homologous recombination or a relatively complex cloning process to construct strains with N-terminal protein tags. Here, we describe a simple CRISPR/Cas9-based method for seamless N-terminal tagging of yeast genes that preserves their endogenous promoter. This method enables the generation of N-terminally tagged strains by introducing an expression vector containing the cas9 gene and a specific gRNA for cleaving the 5′ end of the target gene's protein-coding sequence, along with donor DNA containing the tag sequence and homology arms. gRNA cloning was executed by inverse PCR instead of the conventional method. After verifying the tag, the Cas9 and gRNA expression plasmids were eliminated without using antibiotic-containing medium. By this method, we generated strains that express N-terminally tagged subunits of the TORC1 protein kinase complex and found that these strains are comparable to strains made by conventional methods. Thus, our method provides a cost-effective alternative for seamless N-terminal tagging in baker's yeast.
Pablo Villarreal, Pablo A. Quintrel, Sebastián Olivares‐Muñoz, José J. Ruiz, ,
Published: 18 August 2021
The quest for new wild yeasts has increasingly gained attention because of their potential ability to provide unique organoleptic characters to fermented beverages. In this sense, Patagonia offers a wide diversity of ethanol-tolerant yeasts and stands out as a bioprospecting alternative. This study characterized the genetic and phenotypic diversity of yeast isolates obtained from Central Chilean Patagonia and analyzed their fermentation potential under different fermentative conditions. We recovered 125 colonies from Nothofagus spp. bark samples belonging to five yeast species: Saccharomyces eubayanus, Saccharomyces uvarum, Lachancea cidri, Kregervanrija delftensis and Hanseniaspora valbyensis. High-throughput micro-cultivation assays demonstrated the extensive phenotypic diversity among Patagonian isolates, where Saccharomyces spp and L. cidri isolates exhibited the most outstanding fitness scores across the conditions tested. Fermentation performance assays under wine, mead, and beer conditions demonstrated the specific potential of the different species for each particular beverage. Saccharomyces spp. were the only isolates able to ferment beer wort. Interestingly, we found that L. cidri is a novel candidate species to ferment wine and mead, exceeding the fermentation capacity of a commercial strain. Unlike commercial strains, we found that L. cidri does not require nutritional supplements for efficient mead fermentation. In addition, L. cidri produces succinic and acetic acids, providing a distinct profile to the final fermented product. This work demonstrates the importance of bioprospecting efforts in Patagonia to isolate novel wild yeast strains with extraordinary biotechnological potential for the fermentation industry.
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